Subnanomolar Affinity and Selective Antagonism at α7 Nicotinic Receptor by Combined Modifications of 2-Triethylammonium Ethyl Ether of 4-Stilbenol (MG624)

Modifications of the cationic head and the ethylene linker of 2-(triethylammonium)ethyl ether of 4-stilbenol (MG624) have been proved to produce selective α9*-nAChR antagonism devoid of any effect on the α7-subtype. Here, single structural changes at the styryl portion of MG624 lead to prevailing α7-nAChR antagonism without abolishing α9*-nAChR antagonism. Nevertheless, rigidification of the styryl into an aromatic bicycle, better if including a H-bond donor NH, such as 5-indolyl (31), resulted in higher and more selective α7-nAChR affinity. Hybridization of this modification with the constraint of the 2-triethylammoniumethyloxy portion into (R)-N,N-dimethyl-3-pyrrolidiniumoxy substructure, previously reported as the best modification for the α7-nAChR affinity of MG624 (2), was a winning strategy. The resulting hybrid 33 had a subnanomolar α7-nAChR affinity and was a potent and selective α7-nAChR antagonist, producing at the α7-, but not at the α9*-nAChR, a profound loss of subsequent ACh function.


■ INTRODUCTION
The triethylammonium ethyl ether of 4-stilbenol (1a, MG624) has returned to the fore in very recent years after being reported in the 1950s as a ganglioplegic agent with very weak antimuscarinic activity and no activity on the neuromuscular junction 1,2 and first characterized in 1998 as an antagonist of the homopentameric α7 nicotinic acetylcholine receptors (nAChRs) with moderate and high selectivity, respectively, over the β4and the β2-containing nAChRs. 3 An expanded knowledge of biochemistry, molecular pharmacology, and physiology of nAChR subtypes, along with a number of structure−activity relationship (SAR) studies, has allowed a fuller understanding of MG624′s pharmacological profile and its multifaceted potential as a therapeutic hit. 4−6 Starting from the proven ability of nicotine to promote growth and metastasis of lung tumors by acting on α7and α9α10-nAChRs, we have initially demonstrated that 1a blocks these proproliferative effects on adenocarcinoma cells expressing such nAChRs. 4 We have enlarged the investigation to glioblastoma and to analogues of 1a with elongated O−N alkylene linker, further confirming the antitumor activity and finding that it is greatly advantaged by ethylene bridge lengthening, which generally corresponds to the increasing potency of α7and α9α10-nAChR antagonism. 5 A deeper pharmacological and functional characterization of 1a and its two analogues with tetramethylene and octamethylene O−N linker (1b and 1c, respectively; Chart 1) led us to conclude that, at the α7-nAChRs, they behave as a very weak partial agonist (1a), a silent agonist (1b), and a full antagonist (1c) and that their antiproliferative and cytotoxic effects are not only due to the action on nAChRs. 6 Other nonnicotinic intracellular mechanisms are involved, such as the reduction of the production of mitochondrial and glycolytic adenosine triphosphate (ATP), 5,6 and further studies are needed to understand whether they are independent or cooperative with nicotinic antagonism. Leaving aside the multiple and incompletely defined mechanisms underlying antiproliferative effects (which are therefore hard to interpret), we returned to the electrophysiological assessment of α7and α9α10-nAChR subtypes and a systematic SAR study. We have very recently reported a series of analogues of 1a modified at the ammonium head or at the two-carbon O−N linker. 7 Some of these modifications, detrimental to the α7-nAChR affinity, such as the inclusion of the linker in six-membered nitrogen heterocycles (1e, 1f, and 1g; Chart 1) or oversized increase or decrease of the ammonium head volume (1d and 1h, respectively; Chart 1), led to selective antagonists of human α9α10-nAChR, devoid of any antagonist activity at the α7-nAChR and showing partial agonism at high supramicromolar concentrations. As noted in our recent publication, their selective α9α10-nAChR antagonist activity appeared to consist of opening and rapidly engaging the channel and then blocking it in an open but nonconducting state. These observations are compatible with an open-channel block mechanism, 7 although we emphasize that a definitive demonstration of such a mechanism would require extensive further testing (e.g., competition and voltage-dependence experiments). Among these selective α9α10-nAChR antagonists, the cyclohexyldimethylammonium analogue 1d (Chart 1) stands out for having no α7-nAChR agonist or antagonist effect and very low affinity for the ganglionic α3β4 nicotinic subtype, thus proposing itself as an invaluable tool to define the therapeutic potential of the α9α10-nAChR antagonism. 7 As a second part of the SAR investigation on 1a, we considered modifications at its stilbene scaffold, more specifically at the styryl portion, which represents the distal Chart 1. 1a (MG624) and its Analogues Modified at the Ammonium Ethyl Residue part of such scaffold and whose modifications were expected to be highly influential, as evidenced by the present results, on the interaction with the α7and α9α10-nAChR subtypes. Here, we report the synthesis and the biological evaluation of compounds 3−33 (Chart 2), in which (a) the styryl residue of 1a is totally or partially abolished (3−5), made linear (7), derigidified (6), or further rigidified (8 and 9) also with phenyl bioisosteric replacement (30−32), decorated at phenyl (12− 22) or benzo-condensed (10 and 11), or modified at the vinylene portion by the introduction of heteroatoms and cyclization (23−29) and (b) the two most productive modifications of 1a in terms of the α7-nAChR affinity of this series and of the previous one, 7 respectively, represented by the indolyl analogue 31 and the stilbenoxypyrrolidine 2 (Chart 2), are combined to give hybrid 33. The biological evaluation was performed similarly to that for previously reported analogues of 1a modified at the ammonium ethyl residue. 7 First, an extensive determination of the nAChR subtype binding affinities was performed, followed by the functional screening of a large selection of compounds for α7and α9α10-nAChR antagonisms and then more detailed tests on a few best hits to further study the mechanism of the antagonist activity at the two receptor subtypes. Scheme 1. Reagents and Conditions a a (a) 2-Chloro-N,N-diethylethylamine hydrochloride, K 2 CO 3 , KI, acetone or methyl ethyl ketone, reflux; (b) iodoethane in 1,2-dichloroethane, rt for 3, 23; dichloromethane (DCM), reflux for 4, 5; neat, reflux for 17; EtOH, 70°C for 10, 11; tetrahydrofuran (THF), reflux for 12−14, 16, 18, 67; (c) 1,2-dibromoethane, K 2 CO 3 , KI, methyl ethyl ketone, reflux; (d) NaI, acetone, reflux; (e) diethylamine, toluene, 60°C; (f) benzyl bromide, K 2 CO 3 , acetone, reflux; (g) acetic anhydride, pyridine, rt; (h) methyltriphenylphosphonium bromide, K 2 CO 3 , THF, reflux; (i) 5 M NaOH, THF, 0°C ; (j) appropriate aryl iodide, Pd(OAc) 2 , triethylamine, CH 3 CN, reflux; (k) 1.25 M HCl in MeOH, reflux; (l) triethylamine, toluene, reflux; (m) 1-naphthylmethyltriphenylphosphonium chloride, sodium, EtOH, 10°C to rt; and (n) 2-naphthylmethyltriphenylphosphonium bromide, sodium, EtOH, 10°C to rt. with 2-naphthylmethylenetriphenylphosphorane provided intermediate 70 and its cis isomer, which were separated by chromatography. Successive etherification of 70 with diethylaminoethyl chloride and quaternarization with iodoethane gave 11.
Compound 23 was obtained from hydroquinone by etherification of one hydroxyl with benzyl bromide (38) and of the other with diethylaminoethyl chloride (39), followed by quaternarization of the tertiary amine 39 with iodoethane.
Compounds 6, 7, and 22 were synthesized from trans-4stilbenol, 4-(2-phenylethynyl)phenol, and resveratrol, respectively, according to Scheme 2. Stilbenol was hydrogenated to 4-(2-phenylethyl)phenol (72), O-alkylated with diethylaminoethyl chloride (73), and quaternarized with iodoethane to give 6. 4-(2-Phenylethynyl)phenol was O-alkylated with 1,2dibromoethane (74) and, after bromine/iodine exchange (75), converted to 7 by reaction with triethylamine. Resveratrol was chloroethylated at the 4′-hydroxyl with 1-bromo-2-chloroethane (76) and, after chlorine/iodine exchange (77), converted to 22 by reaction with triethylamine. Scheme 3 shows the syntheses of compounds 8, 9, 25−27, and 30−33. 4-Hydroxyphenyl boronic acid was coupled with 2-bromo-and 1-bromonaphthalene and the resulting intermediates, 78 and 80, respectively, were O-alkylated with diethylaminoethyl chloride (79 and 81) and quaternarized to 8 and 9, respectively, with ethyl iodide. By the same reaction sequence, we synthesized the final compounds 30 and 32 from 4-hydroxyphenyl boronic acid using 6-bromoindole and 5bromobenzofuran respectively. For the synthesis of 31, 4hydroxyphenyl boronic was coupled with N-tosyl-5-bromoindole and the resulting intermediate tosyl amide 96 was hydrolyzed to 97, O-alkylated with diethylaminoethyl chloride (98), and converted to 31 with iodoethane. Intermediate 96 was coupled, by the Mitsunobu reaction, with (R)-N-boc-3hydroxypyrrolidine to give 99. Subsequent reduction with LiAlH 4 provided the N-methyl pyrrolidine 100, which was converted to 33 by treatment with iodomethane. The preparation of 25 and 26 was accomplished from 4benzamidophenol (88)  hydroxybenzanilide (84) was prepared from p-salicylic acid by acetylation (82), conversion into p-hydroxybenzoyl chloride, and reaction with aniline (83) and desacetylation. For the synthesis of 27, phenol was coupled with benzenediazonium salt, generated in situ from aniline, and the obtained compound 92 was reacted with diethylaminoethyl chloride (93) and quaternarized to 27 with iodoethane. Scheme  Biology. Binding Studies. The binding affinities (K i ) of all of the compounds were determined by competition binding experiments on the α7 human subtype, transiently expressed in the SH-SY5Y neuroblastoma cells, 5 and the results are shown in Table 1. With the exception of a few compounds that had a modest affinity for α7-nAChR, competitive binding affinity was also assessed at the human α3β4-nAChR subtype stably transfected in SH-EP1 cells 8 and only select compounds were also tested on the human α4β2-nAChR subtype stably transfected in HEK 293 cells (a generous gift from Dr. Jon Lindstrom 9 ).
We found that, among the compounds modified by structural simplification or rigidification of the styryl residue (3−9), only compounds 5 and 8 had an α7-nAChR K i value close to that of the parent compound 1a (K i = 104 nM) and maintained a modest α7vs α3β4-nAChR and a high α7vs α4β2-nAChR selectivity.
The second set of analogues of 1a, those decorated at the distal phenyl by substituents or an additional condensed benzene (compounds 10−22), showed both a lower affinity for α7-nAChR and reduced selectivity over α3β4-nAChR (where measured).   The same outcome was seen across the third set of analogues (compounds 23−27), those with an ether, an amide, or a diazo linker in place of vinylene. However, replacement of the vinylene linker with an imino linker locked into oxazole or imidazole condensed with the distal phenyl (compounds 28 and 29) led, in the case of benzimidazole 29, to an improved α7-nAChR affinity (K i = 33.6 nM) and α7vs α3β4-nAChR selectivity (10.3 ratio) compared to 1a (K i = 104 nM; α7vs α3β4-nAChR selectivity = 4.2). Among the last set of compounds (30−32), formally derived from 8 by replacement of 2-naphthyl with 5-or 6-indolyl or 5benzoxazolyl, analogous results were obtained for indole 31 (18.7 nM K i and 9.5 ratio).
The binding affinities of the compounds for the α3β4and α7-nAChR subtypes were generally similar or moderately different (≤10-fold ratio), except for the above-mentioned compounds 2 and 33 that had >400-fold preference for α7over α3β4-nAChR and for compound 9, which had ≈30-fold higher affinity for the α3β4than for the α7-nAChR. Approximately half of the compounds were also tested for their affinity for the α4β2-nAChR, and we determined that all had low affinity (K i > 1.2 μM) including compounds 1a, 2, 5, 8, and 33 that we have determined to have high α7-nAChR affinity.
In Vitro Functional Activity on α7 and α9α10-nAChR Subtypes. Compound 1a was earlier shown to be an antagonist of chicken α7-nAChR expressed in Xenopus laevis oocytes (IC 50 = 109 nM) and, more recently, at human α7 and α9α10-nAChR expressed in X. laevis oocytes (IC 50 = 41 and 10 nM, at the respective subtypes). 3,5 Of the compounds reported in this manuscript, nine (6, 25− 31, and 33) were chosen for further testing in functional assays. The selection was driven by the significantly higher α7-nAChR binding potency and selectivity for α7over α3β4- Figure 1. Inhibition concentration response profiles of test compounds at α7or α9α10-nAChR subtypes. mRNA encoding human α7-nAChR subunit was coinjected in X. laevis oocytes along with mRNA for NACHO (in enhanced expression of α7-nAChR; • or ⧫ ). Separate batches of oocytes were injected at a 9:1 ratio with mRNA encoding human α9and α10-nAChR subunits, respectively (open circles). In both cases, the function was tested 1 week after injection, employing two-electrode voltage clamp electrophysiology. Initial stimulations were ACh-only (1 mM, 1 s stimulation, 60 s wash between stimulations, five repeats). These initial stimulations were used to confirm that agonist-alone responses were stabilized, and to provide a positive control, before test compounds were applied. Test compounds were coapplied with ACh stimulations (same 1 mM ACh concentration, 1 s application time, and 60 s wash between applications, as was used for the initial ACh-only stimulations). Concentrations of test compounds were increased from the lowest shown to a maximum of 100 μM in half-log steps. For α7-nAChR, responses were measured in two different ways (as peak currents (•) or as area under the curve ( ⧫ ). In all cases, responses when test compounds were coapplied were normalized to the mean of the magnitude of the final two positive control responses that preceded the introduction of the test compound. Each point is the mean ± standard error of mean (S.E.M.) of five to six responses, with each response being collected from an individual oocyte. Error bars are included for all points but are not visible where the size of the point exceeds that of the corresponding error bars. Even coapplication of compound 6 at 100 μM produced no inhibition of the α7-nAChR function; the resulting data have been omitted to increase clarity. nAChR binding shown by 29 and 31 in comparison with 1a, suggesting further rigidification and introduction of a weakly acidic NH in a suitable position as critical modifications of the styryl moiety of 1a. Therefore, in vitro functional tests were extended also to benzamides 25 and 26, benzoxazole 28, and indole 30, in which one or both the above modifications at the styryl moiety are featured. Compound 33 was selected on the basis of its greatly improved (subnanomolar) α7-nAChR affinity and better α7over α3β4-nAChR selectivity compared to any of the other compounds considered in this study, while the diazo derivative 27 and the phenylethyl analogue 6 were included for the significance of their respective linker modifications. Antagonism of currents activated by 1 mM ACh was determined using X. laevis oocytes that expressed human α7or α9α10-nAChR. Test compounds were coapplied during agonist stimulation. The approach and apparatus were similar to those earlier published for α7-nAChR. 10 However, in this case, α9α10-nAChR was also tested (from oocytes injected with α9 to α10 cRNAs at a 9:1 ratio). As noted in our recent publication, 7 the injection of α9-nAChR cRNA alone produces very little function. In contrast, the injection of our chosen ratio of α9 and α10 cRNA (9:1) produced the most function. Use of this α9:α10 cRNA injection ratio will likely produce functional α9α10-nAChR incorporating subunits in two different stoichiometries: (α9) 2 (α10) 3 and (α9) 3 (α10) 2 . 11 The just noted increase in function following coinjection of the α10 subunit (compared to that if the α9 subunit cRNA is injected alone) further reassures us that the α9-only-nAChR function will be either minimal or absent under the 9:1 α9:α10 cRNA coinjection condition that we use in this and our previous manuscript. We chose to use the same experimental approaches for the present study to allow comparisons to be made to our recently published data. 7 Functional responses of α7-nAChR were assessed using both the measurement of peak currents and net charge gated (area under curve or AUC) to determine whether the rapid-desensitizing property of this subtype at high agonist concentrations might alter the IC 50 values obtained. 12 The concentration response curves thus obtained are shown in Figure 1, and the IC 50 values calculated in each case are summarized in Table 2. Also given in Table 2 are IC 50 values for the lead compound (1a) and for 2 (for comparison since, together with 31, it is the parent compound of 33). As may be seen, in this experiment, under the conditions applied here, IC 50 values calculated using either peak current or AUC measurements of the α7-nAChR function were extremely similar. Despite this, it is worth mentioning that the exceptionally rapid kinetics of the α7-nAChR function at high agonist concentrations raise a concern that the coapplication of antagonists may result in inhibition being measured when drug application is incomplete. For this reason, later parts of this study examined the effects of applying the test compounds by themselves, rather than in a coapplication format.
Except for 6, which had no effect at the α7-subtype, all of the tested compounds were able to inhibit ACh activity at both the subtypes: 27 and 29 with almost identical potency at α7 and α9α10-nAChR, 26 with selectivity toward the α9α10-nAChR, and the remaining 1a, 2, 25, 28, 30, 31, and 33 with higher potency at the α7-nAChR subtype. As shown in Figure 1, some compounds were not able to produce complete inhibition of the nAChR function. In some cases, inhibitory concentration response curves reached a plateau of incomplete antagonism. In others, the maximum test compound concentration of 100 μM was insufficient to produce complete inhibition. However, complete or nearly complete inhibition was observed at both α7and α9α10-nAChR subtypes for 27, 30, 31, and 33. Among these four compounds, 33, the most potent α7-nAChR antagonist of the whole series (1.07 μM IC 50 ), showed the highest α7vs α9α10-nAChR selectivity. Importantly, none of the compounds produced biphasic inhibition of the α9α10-nAChR function. This indicates that in no case do any of the test compounds discriminate between the alternate α9α10-nAChR stoichiometries described in the prior paragraph as likely to be present under the experimental conditions used in this study.
We emphasize here that while sequential applications of test compounds at progressively higher concentrations are common practice, it could result in compounding of effects (in this case, antagonism) produced by previous applications. For this reason, four compounds of special interest were selected to examine their potential intrinsic agonist affinity at α7and α9α10-nAChR and the ability to affect function induced by a subsequent application of an ACh control response. These were compound 6, which exerted no inhibition of α7-nAChR but essentially full inhibition of α9α10-nAChR responses, and compounds 2, 28, and 33, all exhibiting the highest and most selective α7-nAChR antagonist activity (∼1 μM IC 50 , 15−40-fold selectivity over α9α10-nAChR IC 50 ). As for the preceding experiment, repeated applications of ACh (1 mM) were used to ensure the stability of functional responses and define an agonist positive control response. Subsequently, each compound of interest was applied at a single concentration of 100 μM (no ACh present) to oocytes expressing α9α10-nAChR or, excluding 6, to oocytes expressing α7-nAChR. The 100 μM concentration was chosen since it matches the final concentration of the test compounds when they were coapplied with ACh in Figure 1, allowing outcomes to be compared directly. The application of The summary of the test compound antagonist potency (IC 50 values) is derived using the concentration response curves illustrated in Figure 1. Details of the protocols used are given in the Experimental Section and Figure 1 (legend). Please note that IC 50 values at α7-nAChR were calculated using both peak current and area under the curve (AUC) approaches. Both approaches yielded similar values for all compounds tested. Confidence intervals (95% values) are provided in parentheses, which represent the 95% confidence interval of the mean value. "NA", not applicable (i.e., agonist-induced function was not inhibited by the coapplication of the test compound even at 100 μM).
a single concentration, in the absence of ACh, addresses the concern stated at the beginning of this paragraph that sequential applications of the test compounds could result in compounding of their effects. Application of an individual 100 μM pulse of any of compounds 2, 6, 28, or 33 resulted in the partial agonism of α9α10-nAChRs (efficacy of 5−55% of ACh control when responses were measured in terms of the peak current). Interestingly, as previously reported for some analogues of 1a modified at the ammonium ethyl portion, 7 all of the α9α10-nAChR functional responses induced by the test compounds were shorter-lasting than those evoked by the application of the ACh control. When responses were considered in terms of AUC, this resulted in efficacy being reduced to 0.2−3% of ACh control responses. Similar outcomes were found at α7-nAChR for compounds 2, 28, and 33 (15−50% efficacy compared to ACh control). When the intrinsic activity was assessed in terms of AUC, it was reduced somewhat (to 10−22% of the ACh control). This suggests that responses produced at α7-nAChR by the test compound were also somewhat truncated compared to those induced by ACh, albeit to a lesser extent than was seen at α9α10-nAChR. However, compound 6, which did not affect ligand binding at the α7-subtype also, did not have an intrinsic activity at α7-nAChRs ( Figure 2). Of interest, compound 1a has also been reported recently to be an α7-nAChR partial agonist (response to a 100 μM application noted to be ≈40% of the 200 μM ACh control stimulation). 6 Further, at α9α10-nAChR, rebound currents were observed, subsequent to the recovery of the short-duration currents produced in response to the test compound application. These rebound currents lasted longer than initial currents evoked by the test compounds or even preceding the ACh control responses. This phenomenon is illustrated in example traces, shown in Supporting Information Data (pages 20−26). In contrast, at α7-nAChR, only compound 33 evoked a rebound current (example traces are also provided in the same section of Supporting Information Data). Figure 3A illustrates the size of the poststimulation rebound currents evoked by 2, 6, 28, and 33 at α9α10-nAChRs and of 2, 28, and 33 at α7-nAChR. In each case, responses are normalized to the size of control responses previously evoked by ACh positive control responses. Responses are again presented in terms of both peak currents and AUC. As may be seen, peak currents attained during these rebound currents following test compound application varied between 25 and 40% of those produced by ACh control stimulations. However, when AUC was considered, rebound currents varied between 27 and 100% of control. This reflects the effects of the relatively slow onset and recovery of the rebound currents when compared to the initial responses to test compound application.
Responses were also measured for a final ACh (1 mM, 1 s) control stimulation, applied 1 min after test compound application to each oocyte. These final ACh control applications produced a response that was reduced (in some cases, much reduced) in amplitude (whether in terms of peak current or AUC) than the initial ACh control applications. In Figure 3B, we illustrate the residual activities induced by these concluding ACh (1 mM) applications, subsequent to the Figure 2. Partial agonism of human α7or α9α10-nAChRs by compounds 2, 6, 28, or 33 (applied alone). Compounds 2, 6, 28, and 33 were selected (please see the test for criteria) to determine whether they were able to activate human α7or α9α10-nAChR (intrinsic activity). Twoelectrode voltage clamp protocols were similar to those used in Figure 1, including the use of an initial train of ACh (1 mM) control pulses to ensure the stability of responses and collect positive control data for a full agonist. After a further 1 min wash period, compounds of interest were applied for 1 s at 100 μM (the same as the highest concentration applied in Figure 1; in this case, test compounds were applied alone instead of coapplied with ACh). In this case, responses at both α7and α9α10-nAChR were quantified in terms of both peak currents and AUC. For each individual oocyte, and for each method of quantification, responses when test compounds were coapplied were normalized to the mean of the magnitude of the final two positive control responses that preceded the introduction of the test compound. Each bar represents the mean response collected from three individual oocytes, with error bars representing the S.E.M. Points represent responses from individual oocytes. application of compounds 2, 6, 28, and 33 to α9α10-nAChRs or compounds 2, 28, and 33 to α7-nAChRs. As illustrated in Figure 3B, 6 (which has no intrinsic efficacy at α7-nAChR) did, however, significantly block subsequent ACh-induced function at α9α10-nAChRs.
Moving to α7-nAChR responses, compound 33 (which has the highest α7-nAChR affinity and antagonist potency) was the only one in the series to produce an α7-nAChR rebound current ( Figure 3A). This α7-nAChR rebound current induced by 33 was small in terms of peak amplitude, increased slowly, and was very slow to return to baseline. As a result of these slow response kinetics, the intrinsic activity was significantly higher when assessed as AUC than in terms of peak current (57 vs 7%, respectively). Notably, no distinct peak of function was induced by a subsequent control application of ACh; the block of subsequent ACh-induced α7-nAChR activity by compound 33 was thus essentially complete ( Figure 3B). We wished to examine if there was a correlation between the recovery of the rebound currents induced by test compounds at α9α10-nAChR and suppression of the final ACh control stimulation that follows the test compound application. These were calculated, respectively, as "recovery of rebound current" (the percentage by which the rebound current had returned to the prior baseline 1 min following application of the test compound; normalized for each individual oocyte to the peak amplitude of the rebound current over baseline) and "residual ACh-induced current" (i.e., the final ACh control stimulation applied 1 min following the application of the test compound, normalized for each oocyte as a percentage of the amplitude of the mean of the ACh control applications applied before the test compound was applied). Please refer to the Supporting Information Data (page 20) for an illustration of these terms. In our prior publication, we speculated that slow and incomplete recovery of the rebound current preceding the application of the final ACh control pulse could substantially suppress the functional response to the subsequent and final ACh control application. 7 In Figure 4, we plot "residual AChinduced current" (y-axis) against "recovery of rebound current" (x-axis) for the previously published compounds 1d, 1e, 1f, 1g, and 2 along with the new compounds 6, 28, and 33. Please note that, in this figure, only current amplitude data could be used since our previous publication assessed only peak response, and not AUC, values. As can be seen, there is a strong correlation between incomplete recovery of rebound current when the final ACh control pulse is delivered and increased inhibition of ACh-induced currents at α9α10-nAChR. Compounds 2, 28, and 33 showed an almost complete recovery and the highest residual ACh-induced currents, whereas compounds 1d, 1e, and 1f showed a largely incomplete recovery and the lowest residual ACh-induced currents. This confirms what we had speculated in our prior publication, 7 reinforcing the suggestion that the longer the duration of the rebound current (i.e., the slower the disassociation of the compound of interest from the α9α10-nAChR), the greater the suppression of subsequent AChinduced activity is.

■ DISCUSSION
We began by determining the α7-nAChR binding affinity and selectivity over α3β4-nAChR. Regardless of their electronic effects, all of the accomplished modifications of 1a (K i value at α7-nAChR = 104 nM) imply an increase of the steric bulk of the distal phenyl group (compounds 10−22) resulting in significantly lower α7-nAChR affinities. These bulk-increasing modifications also lowered, and sometimes reversed, α7vs α3β4-nAChR selectivity. Such outcomes indicate that the extension of the styryl residue of 1a is a critical issue. Otherwise, within the set of compounds 3−9 (which abolished the distal phenyl or simply varied its positioning), the 2naphthyl analogue 8 showed a profile of α7-nAChR affinities and α7over α3β4-nAChR selectivities very similar to that of 1a. This suggests that the coplanarity of vinylene and phenyl is a requisite of the active conformer of 1a. Among the compounds modified at the vinylene linker (compounds 23− 27), moderate α7-nAChR affinities are shown only by 26 and 27. Notably, these two compounds, having an amide and diazo linker, respectively, maintain the original styryl rigidity (unlike 23 and 24, which have a flexible methyleneoxy linker). Further, unlike the other benzamide 25, compounds 26 and 27 are superimposable to 1a and to its intramolecular cyclized 2naphthyl analogue 8, respectively. These SARs are supported by the subsequent five compounds (28−32), which are all isosteres of 8, thus rigidified analogues of 1a, in which the 2naphthyl of 8 is replaced by a heteroaromatic bicycle without extension with respect to the original styryl moiety but with additional interaction potential due to the presence of heteroatoms. Three of them (28, 30, and 32) show moderate α7-nAChR affinity and the other two, 29 and 31, show high α7-nAChR affinity (33.6 and 18.7 nM K i , respectively). Compared to 1a and 8, benzimidazole 29 and indole 31 have not only significantly higher α7-nAChR affinity but also increased α7vs α3β4-nAChR selectivity. In both, a critical role is played by NH, as indicated by the loss of α7-nAChR affinity resulting from its replacement with O (cf. 29 with 28 and 31 with 32) or its repositioning (cf. 31 with 30). Consistently with all of these observations, a great step forward is achieved by combining the two best modifications of 1a, in terms of α7-nAChR affinity and selectivity, at the stilbene scaffold and at the 2-ammonium ethyl portion, respectively: the replacement of the stilbene scaffold with 4-(5-indolyl)phenyl (compound 31, 18.7 nM K i ) and the previously reported constraint of the 2-ammoniumethyloxy portion into (R)-3-pyrrolidiniumoxy substructure (compound 2, 23 nM K i ). The effects of these two modifications are synergic and the resulting hybrid 33 displays subnanomolar α7-nAChR affinity and very high α7vs α3β4and α4β2-nAChR selectivities. In Chart 3, all of the above structure−affinity relationships are summarized and visualized reproducing, for clarity, the same subdivision of the styryl modifications as in Chart 2 and representing the productive and the unproductive ones compared to 1a in green and red, respectively.
To further elucidate the structural determinants for so high an increase in affinity at the α7-nAChR subtype, the molecular docking of 1a and 33 at the orthosteric binding pocket of the α7α7 dimer extracted and refined from the recently reported cryo-EM structure 7EKP was performed. 13 As shown in Figure   Figure 4. Relationship between residual ACh function after the application of test compounds to α9α10-nAChR and the extent to which the rebound currents that they induce are able to recover before the final ACh application is made (see the text for how these values were calculated). On the Y-axis, the mean amplitude ± S.E.M. of the residual ACh-induced current as % of the previously established ACh control amplitude; on the X-axis, the mean ± S.E.M. recovery of the rebound current as % of the peak rebound current.  where H-bond donors are strongly preferred due to the presence of multiple H-bond acceptors on the target (such as the side chain of Ser-56 or the carbonyl of Glu-184). The additional H-bond network, together with a better fit in the binding pocket, is compatible with the 120 times increase of affinity from 1a to 33.
Also here, as for the previously reported analogues of 1a modified at the ammonium ethyl residue, 7 in vitro functional activity at the α7 and α9α10-nAChRs was determined for a selection of analogues, 9 among the 31 initially tested for binding affinities. As explained above, the selection was centered on benzimidazole 29 and indoles 31 and 33, having the best α7-nAChR profiles; some of their strictest analogues (25, 26, 28, and 30) and, for the representativeness of the vinylene modification, compounds 6 and 27 were then recruited. According to such criteria, as in the previously reported selection of 12 1a analogues modified at the ammonium ethyl residue, 7 compounds with modest or moderate α7-nAChR affinity (see 6,25,26, and 27) were tested for in vitro functional activity as well as compounds with good or high α7-nAChR affinity (28, 29, 30, 31, and 33). It is therefore significant that we obtained, applying selection criteria including a wide range of α7-nAChR affinities in both cases, divergent results for the two series of compounds (those made here vs in the preceding study 7 ). Indeed, among the previously published 1a analogues (those modified at the ethyl ammonium head), we found only compounds unable to produce 100% inhibition of the ACh-induced function at the α7-nAChR or even completely devoid of α7-nAChR antagonism, but all antagonizing ACh activity at α9α10-nAChR. In contrast, among the present 1a analogues modified at the stilbene scaffold in the current study, only one compound, the 4-(2-phenylethyl)phenyl analogue 6, was devoid of α7-nAChR antagonism; all of the other compounds inhibited ACh-induced function at both α7and α9α10-nAChR subtypes and, in the case of the three indolyl analogues 30, 31, and 33, produced 100% inhibition. Notably, compound 6 is, among the selected nine compounds, the one with the poorest α7-nAChR affinity, which could be imputed to the loss of that beneficial coplanarity suggested by the comparison of 8 with 1a. Overall, the modifications at the ethyl ammonium portion of 1a seem effective in impairing the interaction with the α7-nAChR, while α7vs α9α10-nAChR selective antagonism can be achieved only by modifying both the stilbene scaffold and the ethyl ammonium head, as demonstrated by hybrid 33, endowed with subnanomolar α7-nAChR affinity, 100% inhibition of ACh-induced function at the α7-nAChR, and good antagonist selectivity for α7over α9α10-nAChR.

■ CONCLUSIONS
If one considers the results obtained with the present modifications and those previously reported of 1a, one can immediately see that we have found in our prior publication several 1a analogues producing antagonism through a mechanism that we speculated was compatible with the open-channel block at α9α10-nAChR while being completely devoid of α7-nAChR antagonism. In contrast, the present study identified no 1a analogue with the opposite profile (i.e., block of α7-nAChR without α9α10-nAChR antagonism). Against this trend, all of the compounds that behave as antagonists at both the receptor subtypes are more potent at α7than at α9α10-nAChR, except 26 in the present series. However, a marked α7vs α9α10-nAChR-selective antagonism remains elusive. Only 33 shows a significantly selective antagonism at the α7-nAChR together with 100% inhibition of ACh-induced function at both the receptor subtypes. As depicted in Figure 3, it is the only one of the tested compounds that produces a profound loss of subsequent AChinduced function at the α7-nAChR subtype ( Figure 3B) and the only one that also produces a measurable rebound current at this same subtype ( Figure 3A). These features of 33 at α7-nAChR are similar to those that we have described for multiple structurally related (but α9α10-nAChR-selective) antagonists and previously noted to be compatible with an open-channel blocker mechanism. However, as noted in the Introduction section, further experimentation is required to draw a firm conclusion as to the precise mechanism by which these compounds exert antagonism.
Overall, these results show that making the α7and α9α10-nAChR antagonist 1a ineffective on one of the two subtypes or highly subtype-selective is not equally simple in both directions. Single modifications, such as the increase of the ammonium head bulkiness or rigidification of the ethylene linker (1d−1g), 7 but also simple saturation of the vinylene bridge (6) are sufficient to profoundly or completely impair the effects at the α7-nAChR while maintaining the inhibition of ACh function at the α9α10-nAChR. On the other hand, we have not found single modifications of 1a resulting in the exact opposite behavior. However, we were able to obtain a complete loss of residual ACh-induced function at the α7-nAChR while leaving almost unaltered the residual AChinduced function at the other subtype by making modifications at both the portions of 1a, the stilbene and the ethyl ammonium head. These modifications leading to 33 were suggested by the high α7-nAChR affinities of compounds 2 and 31, modified at the ethyl ammonium head and at the stilbene, respectively.
We can thus note that, with regard to 1a modifications, the α9α10-nAChR shows a wider tolerance for structural modifications than the α7-nAChR and this may account for the fact that differentiating α9α10-nAChR antagonism from α7-nAChR antagonism, using 1a as a starting hit, is less difficult than the reverse outcome, for which a finer modulation of the molecular features of the hit is required.
There is a great interest in the physiological roles of α7and α9α10-nAChR and their druggability for the development of optimized therapeutics. 14 To this end, the production of ligands that can reliably discriminate functional effects mediated by α7or α9α10-nAChR is absolutely critical. The identification of 33 and 1d as selective antagonists, at one or the other receptor subtype, having strictly related structures and the same potential mechanism of action, provides a valuable pair of tools and a great aid to future work that will rationally generate new, even-more selective agents. ■ EXPERIMENTAL SECTION Chemistry. All chemicals and solvents were used as received from commercial sources or prepared, as described in the literature. Flash chromatography purifications were performed using KP-Sil 32−63 μm 60 Å cartridges. Thin-layer chromatography (TLC) analyses were carried out on alumina sheets precoated with silica gel 60 F254 and visualized with UV light. The content of saturated aqueous solution of ammonia in eluent mixtures is given as v/v percentage. R f values are given for guidance. 1 H NMR spectra were recorded at 600, 400, 300, or 200 MHz, while 13 C NMR spectra were recorded at 150, 100, or 75 MHz using FT-NMR spectrometers. Chemical shifts are reported in ppm relative to residual solvent (CHCl 3 , MeOH, or DMSO) as the internal standard. Melting points were determined by a Buchi Melting Point B-540 apparatus. Optical rotations were determined using a Jasco P-1010 polarimeter. Liquid chromatography−mass spectrometry (LC−MS) analysis was performed using an Agilent 1200 series solvent delivery system equipped with an autoinjector coupled to a PDA and an Agilent 6400 series triple quadrupole electrospray ionization detector. Gradients of 5% aqueous MeCN + 0.1% HCO 2 H (solvent A), and 95% aqueous MeCN + 0.05% HCO 2 H (solvent B) were employed. Purity was measured by analytical high-performance liquid chromatography (HPLC) on an UltiMate HPLC system (Thermo Scientific) consisting of an LPG-3400A pump (1 mL/min), a WPS-3000SL autosampler, and a DAD-3000D diode array detector using a Gemini-NX C18 column (4.6 mm × 250 mm, 3 μm, 110 Å); gradient elution 0−100% B (MeCN/H 2 O/TFA, 90:10:0.1) in solvent A (H 2 O/TFA, 100:0.1) over 20 min. Data were analyzed using Chromeleon Software v. 6.80. Purity is ≥ 95%, and retention times (R t ) are reported.
Method A. Under a nitrogen atmosphere, a suspension of the appropriate phenol (10 mmol, 1 equiv), K 2 CO 3 (2.0−4 equiv), and KI (0.1 equiv) in the specified solvent (15 mL) was vigorously stirred at reflux temperature for 30 min. The appropriate alkylating agent (1.2−4.2 equiv) was added portionwise or dropwise, and the resulting mixture was refluxed overnight unless specified otherwise. The reaction mixture was cooled to room temperature, and the solid was removed by filtration. The filtrate was concentrated under vacuum, and the crude was purified as specified.  100%).
Method C. All of the solvents used were previously degassed. Under an inert atmosphere, the specified aryl bromide (1.3 mmol, 1 equiv) was dissolved in either 1,2-dimethoxyethane or a mixture toluene/EtOH 1:1 (5 mL). Upon the addition of a solution of Pd(PPh 3 ) 4 (0.35 equiv) in the same solvent (2 mL), the reaction mixture was stirred for 20 min. Afterward, a mixture of EtOH (2 mL)/2 M aq Na 2 CO 3 (4 mL) was added dropwise. When specified, TBAB (0.05 equiv) was also added. A solution of the appropriate boronic acid (1.1 equiv) in 1,2-dimethoxyethane (5 mL) was added dropwise, and the reaction mixture was refluxed overnight. Upon evaporation of the solvent under reduced pressure, the residue was diluted in DCM and filtered through a silica pad, and the solvent was evaporated under reduced pressure. The crude was purified as specified, providing compounds 78, 80, 94, 96, and 101 as oils or solids in moderate to high yields (42−92%).
Method D. The appropriate alkyl halide (2.20 mmol, 1 equiv) was dissolved in a saturated solution of NaI in acetone (10 mL), and the reaction mixture was stirred at reflux temperature overnight. A 10% aqueous solution of Na2S2O5 (20 mL) was added, and the mixture was stirred for 1 h at room temperature. After evaporation of acetone under reduced pressure, the resulting aqueous suspension was extracted with diethyl ether twice. The organic layers were combined and washed with water and then brine. The organic phase was dried over anhydrous Na 2 SO 4 , filtered, and evaporated under reduced pressure. The desired products 36, 44, 62, 63, 75, 77, 86, and 90 were obtained as oils or solids in high yields (72−97%).
Method E. Unless specified otherwise, a solution of the appropriate alkyl iodide (1.35 mmol, 1 equiv) and diethylamine (50 equiv) in toluene (10 mL) was heated at 60°C for 3−4 h. Upon cooling to room temperature, the mixture was washed with water three times. The organic phase was dried over anhydrous Na 2 SO 4 , filtered, and the solvent was evaporated under reduced pressure. The crude was purified as specified, providing the desired compounds 37, 45, 87, and 91 as oils or solids in high yields (84−100%).

Synthesis of 4-Formylphenyl Acetate (40).
A solution of 4hydroxybenzaldehyde (3.00 g, 24.6 mmol, 1 equiv) in pyridine (20 mL) was stirred at 0°C for 30 min. Upon the dropwise addition of acetic anhydride (3.5 mL, 37 mmol, 1.5 equiv) for 30 min, the reaction mixture was warmed to room temperature and stirred until TLC showed full conversion. Afterward, the pH was adjusted to 7 by the dropwise addition of 1 M HCl (10 mL), and the product was extracted in diethyl ether. The combined organic phases were washed with 1 M HCl and 1 M NaOH and then dried over anhydrous Na 2 SO 4 , filtered under reduced pressure, and evaporated, providing the desired product 40 as a pale-yellow oil in an 80% yield. R f = 0.43 (cyclohexane/EtOAc 9:1). 1  Synthesis of . Under an inert atmosphere, methyltriphenylphosphonium bromide (7.16 g, 20.04 mmol, 1 equiv) was added portionwise to a suspension of 4formylphenyl acetate 40 (2.74 g, 16.7 mmol, 1.2 equiv) and K 2 CO 3 (2.76 g, 20.04 mmol, 1.2 equiv) in anhydrous THF (35 mL). The reaction mixture was refluxed for 6 h and then concentrated under reduced pressure. The residue was diluted with diethyl ether and washed with water. The water layer was re-extracted with diethyl ether, and the combined organic phases were dried over anhydrous Na 2 SO 4 , filtered, and evaporated under reduced pressure. The crude was purified by silica gel flash column chromatography (cyclohexane/ EtOAc 95:5), providing the desired compound 41 as a colorless oil in a 62% yield. R f = 0.57 (cyclohexane/EtOAc 9:1). 1  Synthesis of . A solution of 4-vinylphenyl acetate 41 (3.00 g, 18.5 mmol, 1 equiv) in THF (30 mL) was cooled to 0°C. A solution of 5 M NaOH (9 mL, 46.25 mmol, 2.5 equiv) was added dropwise for 5 min, and the reaction mixture was stirred at the same temperature for 4 h. The mixture was quenched for 15 min by the dropwise addition of cold 1.5 M HCl (30 mL) and then further diluted with 60 mL of cold water. The aqueous phase was extracted four times with diethyl ether, and the combined organic phases were dried over anhydrous Na 2 SO 4 , filtered, and concentrated by a rotary evaporator at 25°C. The residue was taken in absolute EtOH (30 mL) and evaporated again at 25°C, providing the desired compound 42 as a solid in a 100% yield. The compound was stored as an ethanolic solution at 0°C to avoid polymerization. Synthesis of 1-(2-Bromoethoxy)- 4-vinylbenzene (43). Obtained from 4-vinylphenol 42 (1.15 g, 9.6 mmol, 1 equiv), K 2 CO 3 (2.5 equiv), KI (0.1 equiv), and dibromoethane (4.2 equiv) in anhydrous methyl ethyl ketone (20 mL), according to Method A, at reflux temperature for 48 h. The residue was resuspended in chloroform (100 mL) and washed with an aqueous solution of 10% NaOH. The organic layer was dried over anhydrous Na 2 SO 4 , filtered, and evaporated under reduced pressure. The crude was purified by silica gel flash column chromatography (cyclohexane/EtOAc 95:5) providing the desired product 43 as a pale-yellow oil in a 55% yield. R f = 0.63 (cyclohexane/EtOAc 9:1). 1
Binding Affinity to α7, α3β4, and α4β2 Nicotinic Receptors. For (±)-[ 3 H]epibatidine (specific activity of 56−60 Ci/mmol; Perkin Elmer, Boston, MA), saturation binding studies were carried out on membrane homogenates. These were prepared from either SH-EP1 cells stably transfected with α3and β4-nAChR subunit cDNAs 8 or HEK 293 cells stably transfected with the α4 and β2 cDNAs (generous gift of Dr. Jon Lindstrom). 9 For saturation experiments, the membrane homogenate aliquots were incubated overnight at 4°C with 0.01−5 nM concentrations of (±)-[ 3 H]epibatidine. Nonspecific binding was determined in parallel by adding 100 nM unlabeled epibatidine (Sigma-Aldrich) to the incubation solutions, as described previously. 33 At the end of the incubation, the samples were filtered on a GFC filter soaked in 0.5% polyethylenimine and washed with 10 mL of ice-cold phosphatebuffered saline (PBS) and the filters were counted in a β counter.
For [ 125 I]-αBungarotoxin ([ 125 I]αBgtx) (specific activity 200−213 Ci/mmol, Perkin Elmer, Boston, MA), saturation binding studies were carried out on a membrane homogenate prepared from SH-SY5Y cells transfected with human α7 cDNA, as described previously. 5 Aliquots of the membrane homogenates were incubated overnight with 0.1−10.0 nM concentrations of [ 125 I]Bgtx at rt. Nonspecific binding was determined in parallel by including in the assay mixture 1 μM of unlabeled αBgtx (Sigma-Aldrich). After incubation, the samples were filtered as described for (±)-[ 3 H]epibatidine binding.
For competition studies, the inhibition of [ 3 H]epibatidine and [ 125 I] αBgtx binding was measured by incubating the membranes transfected with the appropriate subtype with increasing concentrations of the compounds (1 nM to 1 mM) 5 min followed by overnight incubation at 4°C, with 0.1 nM of [ 3 H]epibatidine for the α4β2 subtype or 0.25 nM of [ 3 H]epibatidine for the α3β4 subtype or at rt with 2−3 nM of [ 125 I]αBgtx in the case of the α7-subtype. At the end of the incubation time, the samples were processed as described for the saturation studies.
[ 3 H]epibatidine binding was determined by liquid scintillation counting in a β counter, and [ 125 I] αBgtx binding was determined by direct counting in a γ counter. Saturation binding data were evaluated by one-site competitive binding curve-fitting procedures using GraphPad Prism version 6 (GraphPad Software, CA). In the saturation binding assay, the maximum specific binding (B max ) and the equilibrium binding constant (K d ) values were calculated using one-site�specific binding with the Hill slope�model. K i values were obtained by fitting three independent competition binding experiments, each performed in duplicate for each compound on each subtype. Inhibition constants (K i ) were estimated by reference to the K d of the radioligand, according to the Cheng−Prusoff equation and are expressed as nM values.
Two-Electrode Voltage Clamp (TEVC) Recording of α7-and α9α10-nAChR Functions. For functional pharmacology studies, two-electrode voltage clamp recordings were performed, using human nAChR subunits heterologously expressed in X. laevis oocytes. Approaches were closely related to those previously detailed. 10 Briefly, X. laevis oocytes were purchased from Ecocyte Bioscience US (Austin, TX), and the incubation temperature was 13°C. Harvesting of oocytes from X. laevis by EcoCyte follows the guidelines of the National Institute of Health's Office of Laboratory Animal Welfare and was authorized under IACUC number #1019-1 (valid through December 2022). Injections of nAChR subunit mRNA were made using glass micropipettes (outer diameter ≈40 μm, resistance 2−6 MΩ), and mRNA was injected in a total volume of 40 nL. For α7-nAChR, 1.25 ng of α7-nAChR subunit mRNA was injected per oocyte along with 0.125 ng of NACHO mRNA to improve functional expression. 34 For α9α10-nAChR, a total of 10 ng of nAChR subunit mRNA was injected using α9 to α10 cRNAs in a 9:1 ratio by mass.
TEVC recordings were made in oocyte saline solution (82.5 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 1.8 mM CaCl 2 · 2 H 2 O, and 1 mM MgCl 2 · 6 H 2 O, pH 7.4) and were performed at room temperature (20°C ). One week after injection, oocytes were voltage-clamped (−70 mV; Axoclamp 900A amplifier, Molecular Devices, Sunnyvale, CA). Recordings were sampled at 10 kHz (low-pass Bessel filter, 40 Hz; high-pass filter, DC) and saved to disk (Clampex v10.2; Molecular Devices). To ensure the quality of recordings, oocytes with leak currents (I leak ) > 50 nA were discarded without being recorded. In all cases, initial control stimulations (ACh, 1 mM, applied for 1 s) were performed, with a 60 s washout (no drug) between control stimulations (total of five stimulations). This allowed us to define a 100% response control and to ascertain that run-down or desensitization was not occurring due to repeated ACh stimulation.
For antagonist concentration response curves, test compounds were applied simultaneously with 1 mM ACh, starting with the lowest concentration of the test compound and increasing in half-log steps to a maximum concentration of 100 μM. The standard 1 min spacing between stimulation was maintained. Data for each oocyte were normalized by expressing the peak function in the presence of test compounds as % of the control function (the mean peak function measured across the initial control stimulations was defined as 100% for each oocyte). IC 50 values were calculated from these normalized nAChR-mediated currents through nonlinear least-squares curve fitting (GraphPad Prism 5.0; GraphPad Software, Inc., La Jolla, CA).
The intrinsic agonist efficacy of test compounds was measured by applying them (alone at 100 μM, 1 s application time, no ACh coapplication) 1 min following the last initial control stimulation. The peak function following the addition of the test compound was normalized for each oocyte in the same way just described for antagonist concentration curves. The same normalization was applied to the peak of any rebound current observed during the 60 s washout period following the application of the test compound and to the peak function induced by a final control application of ACh (1 mM, 1 s application time).
Computational Modeling. Compounds 1a and 33 were drawn with the two-dimensional (2D) sketch editor of Maestro and prepared for docking using Ligprep, with default settings. The dimeric α7α7 interface containing EVP-6124 was extracted from the cryo-EM of the full-length structure of the human α7-nAChR (7EKP) and prepared with the Protein Preparation Wizard according to default settings. Compound 33 was docked using the Induced Fit Protocol of Schrodinger, 35 selecting the current ligand (EVP-6124) as the docking centroid, Glide XP redocking, and a scaling factor of 1.0, to avoid excessive deformation of the binding site. The best-scoring pose according to the IFD score and the XP GScore also respected the best-known conserved ligand−α7-nAChR interaction, by placing the positively charged nitrogen within the aromatic box and was therefore selected. Compound 1a was docked using Glide XP docking with default settings, with a grid centered on ligand 33, and the bestscored pose according to the XP GScore was selected. The binding site analysis was performed using Sitemap, centered on 33 and default settings.

* sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c01256. 1 H NMR and 13 C NMR of the final compounds; HPLC traces of key final compounds (6, 28, and 33); example traces of two-electrode voltage clamp recordings (PDF) Molecular formula strings (CSV)